Fluorescence resonance energy transfer (FRET) as a high-throughput assay for coupling reactions. Arylation of amines as a case study

Article abstract of DOI:10.1021/ja034161p

A solution-phase assay based on fluorescence resonance energy transfer (FRET) was developed for high-throughput screening of palladium catalyzed aminations of aryl halides. Dansylpiperazine was used as the fluorescent component and a chloro- or bromoarene tagged with an azodye as the quenching partner. Fluorescence intensities of reaction aliquots correlated linearly with reaction yield after dilution to appropriate concentrations. A library of 119 phosphine and heterocyclic carbene ligands was evaluated in duplicate reactions of two combinations. In general, the FRET assay displayed excellent reproducibility, with less than 5% of the duplicate experiments showing significant variability in yields. Among reactions producing greater than 50% yield, the average percent uncertainty was just 5%. For a small subset of sterically hindered ligands, differences in yields between 10 and 20% were observed between the substrates bearing dyes for the FRET assay and substrates that are unfunctionalized. However, the remaining catalyst combinations gave yields similar to those expected from literature precedent. In addition to an evaluation of the accuracy of the FRET assay, this work includes the use of the FRET assay to investigate relative activities of various catalysts for the amination of aryl bromides and chlorides and to find conditions for aminations in more polar solvents. Reactions with K₃PO₄ base in aqueous mixtures of polar and nonpolar organic solvents were shown to be appropriate for the amination chemistry.

Full text of DOI:10.1021/ja034161p

Published on Web 05/17/2003
Fluorescence Resonance Energy Transfer (FRET) as a
High-Throughput Assay for Coupling Reactions. Arylation of
Amines as a Case Study
Shaun R. Stauffer and John F. Hartwig*
Contribution from the Department of Chemistry, Yale UniVersity, P.O. Box 208107,
New HaVen, Connecticut 06520-8107
Received January 14, 2003; E-mail: John.Hartwig@yale.edu
Abstract: A solution-phase assay based on fluorescence resonance energy transfer (FRET) was developed
for high-throughput screening of palladium catalyzed aminations of aryl halides. Dansylpiperazine was used
as the fluorescent component and a chloro- or bromoarene tagged with an azodye as the quenching partner.
Fluorescence intensities of reaction aliquots correlated linearly with reaction yield after dilution to appropriate
concentrations. A library of 119 phosphine and heterocyclic carbene ligands was evaluated in duplicate
reactions of two combinations. In general, the FRET assay displayed excellent reproducibility, with less
than 5% of the duplicate experiments showing significant variability in yields. Among reactions producing
greater than 50% yield, the average percent uncertainty was just 5%. For a small subset of sterically hindered
ligands, differences in yields between 10 and 20% were observed between the substrates bearing dyes
for the FRET assay and substrates that are unfunctionalized. However, the remaining catalyst combinations
gave yields similar to those expected from literature precedent. In addition to an evaluation of the accuracy
of the FRET assay, this work includes the use of the FRET assay to investigate relative activities of various
catalysts for the amination of aryl bromides and chlorides and to find conditions for aminations in more
polar solvents. Reactions with K3PO4 base in aqueous mixtures of polar and nonpolar organic solvents
were shown to be appropriate for the amination chemistry.
Introduction
analyze catalyst activity. Each method has its attributes, but none
of them applies to all problems. Each is slower than desired,1,2
The challenge of pinpointing the optimal catalysts for a
particular reaction or generating an initial catalyst structure for
studies of new processes has created the desire for methods to
screen catalyst activity more rapidly. Several approaches to
address this issue have emerged, and they provide the potential
to accelerate the discovery of new reactions. For example,
HPLC,1,2 mass spectrometry,3-6 colorimetric assays,7,8 IR
thermography,9-11 capillary electrophoresis,12,13 and fluores-
cence14-16 have all been used in a high-throughput fashion to
equipment intensive,3-6,12,13 or narrow in the scope of reaction
that can be analyzed.7,8 Some analyze overall activity and do
not probe directly the formation of a desired material.9-11 We
envisioned a rapid and general assay to screen for product
formation that would use inexpensive, currently available
equipment and would be highly sensitive, noninvasive, and time
resolved.
To address this goal, we have developed a method to monitor
product formation by the phenomenon of fluorescence resonance
energy transfer (FRET).17,18 We have applied this method to
the analysis of three cross-coupling processes. The applications
of this method to two of these processes have recently been
communicated.19,20 In one case, we uncovered conditions for
the first general arylation of cyanoacetates. In the second, we
uncovered conditions for the first Heck coupling of unactivated
bromoarenes at room temperature.21 We now describe the scope
(1) The sensitivity of small-scale catalyst screens to the ratio of ligand to metal
has been noted previously: Porte, A. M.; Reibenspies, J.; Burgess, K. J.
Am. Chem. Soc. 1998, 120, 9180-9187.
(2) Burgess, K.; Lim, H.-J.; Porte, A. M.; Sulikowski, G. A. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 220-222.
(3) Hinderling, C.; Chen, P. Angew. Chem., Int. Ed. 1999, 38, 2253-2256.
(4) Senkan, S.; Krantz, K.; Ozturk, S.; Zengin, V.; Onal, I. Angew. Chem.,
Int. Ed. 1999, 38, 2794-2799.
(5) Reetz, M. T.; Becker, m. H.; Klein, H.-W.; Stockigt, D. Angew. Chem.,
Int. Ed. 1999, 38, 1758-1761.
(6) Guo, J.; Wu, J.; Siuzdak, G.; Finn, M. G. Angew. Chem., Int. Ed. 1999,
38, 1755-1758.
(7) Lavastre, O.; Morken, J. P. Angew. Chem., Int. Ed. 1999, 38, 3163-3165.
(8) Cooper, A. C.; McAlexander, L. H.; Lee, D.-H.; Torres, M. T.; Crabtree,
R. H. J. Am. Chem. Soc. 1998, 120, 9971-9972.
(14) Shaughnessy, K. H.; Hartwig, J. F. J. Am. Chem. Soc. 1999, 121, 2123-
2132.
(15) Copeland, G. T.; Miller, S. J. J. Am. Chem. Soc. 1999, 121, 4306-4307.
(16) Harris, R. F.; Nation, A. J.; Copeland, G. T.; Miller, S. J. J. Am. Chem.
Soc. 2000, 122, 11270-11271.
(9) Reetz, M. T.; Becker, M. H.; Liebl, M.; Furstner, A. Angew. Chem., Int.
Ed. 2000, 39, 1236-1239.
(10) Taylor, S. J.; Morken, J. P. Science 1998, 280, 267-270.
(11) Holzwarth, A.; Schmidt, H.-W.; Maier, W. F. Angew. Chem., Int. Ed. 1998,
37, 2644-2650.
(17) Stryer, L.; Haugland, R. P. Proc. Natl. Acad. Sci. U.S.A. 1967, 58, 719-
726.
(18) Wu, P.; Brand, L. Anal. Biochem. 1994, 218, 1-13.
(19) Stauffer, S. R.; Beare, N. F.; Stambuli, J. P.; Hartwig, J. F. J. Am. Chem.
Soc. 2001, 123, 4641-4642.
(12) Zhang, Y.; Gong, X.; Zhang, H.; Larock, R. C.; Yeung, E. S. J. Comb.
Chem. 2000, 2, 450-452.
(13) Reetz, M. T.; Kuhling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Angew.
Chem., Int. Ed. 2000, 39, 3891-3893.
(20) Stambuli, J. P.; Stauffer, S. R.; Shaughnessy, K. H.; Hartwig, J. F. J. Am.
Chem. Soc. 2001, 123, 2677-2678.
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A R T I C L E S
Stauffer and Hartwig
Figure 1. General approach to using FRET for analyzing the formation of
covalent bonds.
Figure 2. FRET pair chosen for derivatization with substrates for the
amination chemistry.
and limitations of this FRET-based assay for the palladium-
catalyzed amination of aryl halides. A large number of ligands
have already been screened for this reaction, and these previous
results provide a framework for evaluating the accuracy of
reaction yields obtained by the FRET method.
In addition to evaluating the accuracy of the FRET assay by
studying the amination of aryl halides, we hoped to reveal
whether the order of reactivity of catalysts containing different
ligands depended on the identity of the halogen in the aryl halide
and whether high yields could be obtained with bases weaker
than NaOt-Bu in media more polar than arenes and ethers that
are most commonly used for the amination process. The
turnover-limiting step and reactions that deactivate the catalyst
can depend on the halide and create different trends in catalyst
reactivity for aryl chlorides, bromides, and iodides.22,23 The use
of weaker bases in more polar media would increase the scope
of the amination, particulary with pharmacuetical intermediates
that are often insoluble in aromatic and ether solvents.
General Strategy. To develop an assay that met the various
criteria outlined in the previous section, we built upon a
qualitative fluorescent method we published previously.14 The
previous method involved reactions of one reagent supported
on polystyrene beads with a second reagent that was conve-
niently tagged with a fluorophore. This method provided a
qualitative, rather than quantitative, assessment of catalytic
activity and required isolation and washing of the resin-bound
product. The modification of this method to generate a quantita-
tive assay could permit the determination of kinetic parameters
for multiple substrates in a parallel fashion. To develop such a
quantitative assay, we changed the reaction partner from a
reagent on a solid support to a reagent that would quench
fluorescence by FRET.
Figure 3. Amine substrate 3 used in the amination studies.
fluorescence from B is generated by this energy transfer if the
acceptor attached to B is emissive.
The relative emission intensity can then be converted to
reaction yield by the negative linear relationship between
emission intensity and mole fraction of coupled product within
an appropriate range of concentrations. An inexpensive, com-
mercial fluorescence plate reader would require only 1 s per
sample to measure the fluorescence intensities. The FRET assay
could be miniaturized to evaluate sub-micromolar quantities of
substrate per well and even less catalyst.
Design of Chromophoric Substrates. The two chro-
mophores chosen for the FRET assay are shown in Figure 2.
The dansyl group was selected as the fluorophore. It is
inexpensive and possesses functional groups that are inert toward
most types of coupling reactions. This chromophore can be
attached to different reactants through the sulfonyl group and
has a large Stokes’ shift (∼130 nm). An azodye was chosen as
the acceptor molecule because it has an absorption band that
overlaps with the fluorescence emission maximum of the dansyl
group. The particular azodye selected for this study contains
an ortho substituent to discourage coordination of the azo-
nitrogens and an amino functionality to tether reactant B. A
large number of nucleophiles and electrophiles could be attached
to this FRET pair for screening of a variety of reactions.
Results and Discussion
FRET has been used over the past four decades as a versatile
method for measuring noncovalent binding events in biological
and macromolecular systems. FRET takes place when the
fluorescence emission band of one molecule (donor) overlaps
with an excitation band of a second (acceptor) that is within
20-80 Å of the donor.17,18 At an appropriate constant total
concentration of free and associated FRET pairs, the emission
of the FRET donor is inversely proportional to the mole fraction
of associated molecules.
Selection and Synthesis of the Chromophores. A method
to analyze palladium-catalyzed arylation of amines with FRET
requires a dye and quencher that are stable to the basic
conditions of the amination process. The dansyl sulfonyl
functionality was attached to a piperazine to form dansyl
derivative 3 in Figure 3. Piperazines participate in aminations
of aryl halides,24-27 and the sulfonamide should be stable to
the tert-butoxide base in organic solvents.28
We used FRET as a quantitative probe for the formation of
a desired covalent bond in a reaction product by the scenario
in Figure 1. The coupling of a reactant A that is tethered to a
donor fluorophore and a reactant B that is tethered to an acceptor
dye or quencher could alter the fluorescence properties of the
solution. If there is sufficient spectral overlap between the
fluorophore and acceptor and a short enough distance between
them, then energy transfer will take place. In this case, the
fluorescence from A is quenched by the acceptor B. In addition,
Several methods for tethering aryl halides to azodye quench-
ers were explored (Scheme 1, 4-7). Scheme 1 shows the four
modified dyes examined for the arylation of 3 at 80 °C with
(23) Littke, A.; Dai, C.; Fu, G. J. Am. Chem. Soc. 2000, 122, 4020-4028.
(24) Zhao, S.; Miller, A. K.; Berger, J.; Flippin, L. A. Tetrahedron Lett. 1996,
37, 4463-4466.
(25) Nishiyama, M.; Yamamoto, T.; Koie, Y. Tetrahedron Lett. 1998, 39, 617-
620.
(26) Lo´pez-Rodriguez, M.; Viso, A.; Benhamu´, B.; Rominguera, J.; Murcia,
M. Bioorg. Med. Chem. Lett. 1999, 9, 2339-2342.
(27) Hepperle, M.; Eckert, J.; Gala, D.; Shen, L.; Evans, C. A.; Goodman, A.
Tetrahedron Lett. 2002, 43, 3359-3363.
(21) Littke, A. F.; Fu, G. C. J. Am. Chem. Soc. 2001, 123, 6989-7000.
(22) Beare, N. A.; Hartwig, J. F. Unpublished results.
(28) Greene, T. W.; Wuts, P. G. M. In ProtectiVe Groups in Organic Synthesis,
3rd ed.; John Wiley and Sons: New York, 1999; p 616.
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FRET as a High-Throughput Assay for Coupling Reactions
A R T I C L E S
Scheme 1
Scheme 2 a
NaOt-Bu as base and the combination of 3 mol % Pd(OAc)2
and 2.8 mol % (t-Bu)3P as catalyst.29
Modified dyes 4-6 underwent partial to complete cleavage
of a carbon-carbon bond of the tether in competition with the
formation of coupled product. Substrate 4 underwent deben-
zylation, and substrates 5 and 6 underwent C-C cleavage to
generate an N-methyl arylamine and an N-ethyl, N-methyl
arylamine, respectively. Isolated yields for the coupling process
were roughly 50%, and isolated yields of the fragmented dye
were in the range of 40%. No products from side reactions of
the dansylamine were observed.
a Reagents and conditions: (a) (i) n-BuLi, -75 °C; (ii) 4-ClC6H4CH2Cl
or 4-BrC6H4CH2Br; (iii) HCl; (b) BH3-SMe2; (c) TsCl, Et3N; (d) (i)
LiNHAr (Ar ) azodye); (ii) NaHMDS, Boc2O.
The mechanism for these fragmentations is unclear. The
products from amination of 5 and 6 were subjected to the
reaction conditions after they were isolated in pure form, and
no cleavage products were detected. Thus, the cleavage occurs
with the starting material or a reaction intermediate and not with
coupled product.
In contrast, Boc-substituted, propyl-linked 7 produced the
coupled product 8 without side reactions. This substrate formed
the product of aryl halide amination in 70% isolated yield under
the previous conditions. Thus, we used this substrate and its
chloride analogue for all further studies. Substrate 7 was
prepared by a straightforward sequence starting from 2,4,4-
trimethyl-2-oxazoline and an appropriate benzyl halide, as
shown in Scheme 2. By this reaction sequence, we produced
multiple grams of the bromo- and chloroarene substrates 7 and
9 in five steps.
Figure 4. Mole fraction of product versus fluorescence intensity (λexcitation
/
λemmision ) 360 nm/460 nm) for solutions of product 8, fluorophore 3, and
quencher 7 at a total concentration of 10-5 M containing 0.0, 0.2, 0.4, 0.6,
0.8, and 1.0 mol fraction of 8.
Evaluation of Changes in Emission Intensity upon Cou-
pling. To generate a calibration curve for determining reaction
yields, we obtained the data summarized in Figure 4. Solutions
containing various ratios of coupled product 8 and starting
materials 3 and 7 (1:1 ratio) were prepared, and the fluorescence
intensities of the solutions were obtained with a fluorescence
plate reader. Correlation coefficients for such plots were
typically 0.99. These plots were used to determine percent yield
from the fluorescence intensity of aliquots removed from the
amination reactions (see Supporting Information for further
details).
Library of Dative Ligands. Concurrent with the development
of the FRET assay, we expanded our library of ancillary ligands.
We assembled an array of pure ligands that contained repre-
sentatives from families that spanned a broad spectrum of both
steric and electronic properties. New structures were based on
(29) Hartwig, J. F.; Kawatsura, M.; Hauck, S. I.; Shaughnessy, K. H.; Alcazar-
Roman, L. M. J. Org. Chem. 1999, 64, 5575-5580.
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A R T I C L E S
Stauffer and Hartwig
Figure 5. General structural classes contained in the ligand library.
recent advances in ligand design.29-34 Some of the more
prevalent families of ligands in the library are sumarized in
Figure 5. The structures of each member of the 119-member
library are provided as Supporting Information. One-third of
the ligands were obtained from commercial sources, one-third
were prepared according to literature procedures, and the
remaining 39 were new structures.19,20 Of the 39 new ligands,
classes that could be prepared by a divergent synthesis were
explored most widely. These classes included ferrocenyl,
biphenyl, and alkyl phosphines.
aryl bromides observed during previous studies. For example,
reactions with catalysts bearing Tol-BINAP or bis(di-tert-
butylphosphino)ferrocene were found to generate coupled
product after 9 h at 60 °C in greater than 70% yield, as
determined by FRET, with a standard deviation less than 5%.
These data are consistent with literature data obtained by
conventional methods for reactions with these pairs of ligands
and substrates.29,36 Moreover, reactions catalyzed by complexes
of ligands, such as tri(o-tolyl)phosphine and dppe that are known
to generate catalysts with low activity at room temperature,37,38
gave yields less than 20%, as determined by FRET.
Conditions and Reproducibility of Screening the Reactions
of Dansylpiperazine with Bromo- and Chloroarene Dyes 7
and 9. Yields of reactions of the tagged substrates determined
by FRET were compared to yields of reactions of related
untagged substrates determined by conventional methods. Reac-
tions of the tagged substrates were conducted initially with 12
different ligands in 0.5 dram screw-top vials. The reaction
vessels contained 15 µmol of amine 3 and bromide 7, 3 mol %
ligand and 3 mol % Pd(OAc)2, and 1.5 equiv of NaOt-Bu in
approximately 100 µL (0.14 M) of solvent. Reactions were
assembled in a drybox, and all reagents were added by pipet
from stock solutions. The vials were placed in an aluminum
block, heated at 60 °C, and agitated with a STEM shaker.
After 9 h, an aliquot was removed from each vial and diluted
to 10-5 M with m-xylene. The fluorescence intensities of the
samples were measured and converted to percent yield with a
curve, such as that shown in Figure 4. The standard deviation
in yield determined by fluoresence ranged from 1 to 8% (n g
3) for the 12 model reactions conducted three or more times.
In general, reactions with catalysts that produced coupled
product in less than 20% yield displayed the greatest uncertainty,
while reactions with catalysts that produced coupled product
in high yields displayed good reproducibility.35
The reactions were then conducted in a Zinsser 96-well glass
plate. A plate with 45° corners and 300 µL wells sandwiched
between two aluminum plates (see Experimental Section for
details) was used to conduct reactions with 50 µL volumes. With
the sensitive fluorescence detection method and appropriate
reaction blocks, the reactions could be further reduced in
volume, and reagents could be added with standard robotic liquid
dispensors.
With this methodology, the 119-member library of ligands
was evaluated with the bromide 7 and chloride 9 under a variety
of reaction conditions. For the broader screening in 96-well
plates, CpPd(allyl) was used instead of Pd(OAc)2 as catalyst
precursor because of its enhanced solubility. Depending on the
batch, Pd(OAc)2 precipitated from dioxane within 15-30 min
at room temperature; however, Pd(OAc)2 was utilized with
certain ligands (vide infra). Palladium(0) precursors, such as
Pdn(dba)m (n ) 1, 2; m ) 2, 3), were not soluble enough to
generate stock solutions that would allow for a final substrate
concentration above 0.1 M. To minimize variability in catalyst
incubation time, the ligand stocks were added first, and the
palladium source was added just before addition of the base.
Stock solutions of CpPd(allyl), fluorophore, and NaOt-Bu were
stored no longer than 1 day at -30 °C in a drybox. Stock
solutions of the ligand library and reactants 7 and 9 were stored
for several weeks at -30 °C in a drybox.
The trends in reaction yields were consistent with the relative
activity of these catalysts for reactions of secondary amines with
(30) Aranyos, A.; Old, D. W.; Kiyomori, A.; Wolfe, J. P.; Sadighi, J. P.;
Buchwald, S. L. J. Am. Chem. Soc. 1999, 121, 4369-4378.
CpPd(allyl) is a soluble catalyst precursor that is readily
reduced to common Pd(0) complexes.39,40 Free phosphine
(31) Zhang, C. M.; Huang, J. K.; Trudell, M. L.; Nolan, S. P. J. Am. Chem.
Soc. 2000, 122, 12051-12052.
(32) Bei, X.; Guram, A. S.; Turner, H. W.; Weinberg, W. H. Tetrahedron Lett.
1999, 40, 1237-1240.
(36) Wolfe, J. P.; Buchwald, S. L. J. Org. Chem. 2000, 65, 1144-1157.
(37) Guram, A. S.; Rennels, R. A.; Buchwald, S. L. Angew. Chem., Int. Ed.
Engl. 1995, 34, 1348-1350.
(33) Hamann, B. C.; Hartwig, J. F. J. Am. Chem. Soc. 1998, 120, 7369-7370.
(34) van Strijdonck, G. P. F.; Boele, M. D. K.; Kamer, P. C. J.; de Vries, J. G.;
van Leeuwen, P. W. N. M. Eur. J. Inorg. Chem. 1999, 1073-1076.
(35) A similar larger variation in reactions that occurred in lower yields was
noted in a study on copper-catalyzed chemistry: Fagan, P. J.; Hauptman,
E.; Shapiro, R.; Casalnuovo, A. J. Am. Chem. Soc. 2000, 122, 5043-5051.
(38) Louie, J.; Hartwig, J. F. Tetrahedron Lett. 1995, 36, 3609-3612.
(39) Wallow, T. I.; Goodson, F. E.; Novak, B. M. Organometallics 1996, 15,
3708-3716.
(40) Yoshida, T.; Otsuka, S. Inorg. Synth. 1985, 28, 113-119.
9
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FRET as a High-Throughput Assay for Coupling Reactions
A R T I C L E S
accord with studies conducted over the past few years that have
generatedhighlyactivecatalystsforcross-couplingprocesses.21,23,25,33,44-49
These studies have shown that reaction rates for aryl halide
aminations catalyzed by complexes of sterically hindered alkyl
phosphines are much faster than those catalyzed by complexes
of arylphosphines. The most effective phosphines for reactions
of the two dyes at room temperature contained a ferrocenyl (L4-
L21), a biphenyl (L27-L38), or a third alkyl (L69-L76) group
in addition to one or two tert-butyl groups.
The most effective imidazolium or dihydroimidazolium ligand
precursors contained hindered 2,6-diisopropylphenyl groups on
nitrogen. These results were also consistent with the high activity
of sterically hindered ligands. These results led us to test the
dihydroimidazolium salts for a variety of aryl halide amination
reactions. The scope of room-temperature aminations catalyzed
by palladium and this ligand precursor was published recently.41
Nolan has also investigated the activity of this ligand for
aminations at elevated temperatures,50 for related arylations of
carbonyl compounds,51 and for more classic couplings.52-54
Figure 6. Yields determined by fluorescence measurements for the coupling
of 3 with 7 in the presence of palladium catalysts containing 119 different
ligands.
ligands induce reduction of CpPd(allyl) to Pd(0) phosphine
complexes.40 However, reactions of imidazolium and dihy-
droimidazolium salts with CpPd(allyl) may form the Pd(0)
complexes slowly or in low yield because the ligand was added
in its protonated form and base was added after the palladium.
Indeed, reactions catalyzed by imidazolium and dihydroimida-
zolium salts in combination with (allyl)PdCp occurred in yields
below 30%, but reactions catalyzed by these ligand precursors
and Pd(OAc)2 occurred in yields over 80%. Thus, we tested in
parallel both CpPd(allyl) and Pd(OAc)2 as precursors for
reactions with the imidazolium and dihydroimidazolium salts.41,42
Figure 6 depicts the average percent yields deduced from
fluorescence intensity measurements for duplicate reactions with
each member of the ligand library. All reactions were conducted
for 16 h at room temperature with fluorophore 3 and bromoarene
7 (see Supporting Information for complete details, including
statistical variation). Results from duplicate runs indicated
excellent reproducibility of the yields obtained by the fluores-
cence method from reactions that occurred in moderate to high
yields. In the set of 119 duplicate reactions, only 16 reactions
showed a percent uncertainty43 greater than 30, and of these 16
reactions, only three (L15, L28, and L66) occurred in greater
than 40% yield in either run. Thus, only 3 of the 119 reactions
showed significant variation in cases that could have synthetic
value. Moreover, analysis of a second aliquot removed from
the second run of these three reactions indicated the formation
of coupled product in yields that were within 30% of those
obtained from the first run of these three reactions. Thus, an
error in dilution of the first aliquot taken from the second run
with these three ligands accounts for the larger variation in yield.
This type of error could be recognized in all cases, as long as
the experiment is conducted in duplicate.
Although the differences in activities were small between
derivatives of the pentaphenylferrocenyl phosphines (Q-phos)55
bearing methoxy, hydrogen, and trifluoromethyl groups at the
para-position of the aryl rings, some trends could be assigned.
The Q-phos derivative bearing electron-withdrawing trifluo-
romethyl groups (L19 in Figure 7) appeared to generate catalysts
that were more active toward bromoarenes than those lacking
this group or bearing electron-donating groups. Consistent with
this result from the FRET experiments, reactions of 0.5 mmol
bromotoluene with morpholine at room temperature catalyzed
by 3% Pd(dba)2 and 3% L19 occurred 2-3 times faster and to
higher conversions after 48 h than the same reaction catalyzed
by 3% Pd(dba)2 and 3% parent Q-phos ligand L17.
The catalyst generated from Xantphos L100, first reported
by van Leeuwen,56 was the only one that lacked alkyl groups
and formed over 50% yield of coupled product after 16 h at
room temperature. In contrast, ligand L64, the bis(di-tert-butyl)
analogue of L100, was only moderately effective for the test
reaction (37 ( 9% yield). The more commonly used catalysts
containing BINAP36 and DPPF57 showed little or no activity
for reactions of aryl bromides at room temperature.58
Two false positives emerged from this screen. Catalysts
derived from ligands L59 and L106 gave yields that were
(44) Watanabe, M.; Nishiyama, M.; Koie, Y. Tetrahedron Lett. 1999, 40, 8837-
8840.
(45) Littke, A. F.; Fu, G. C. Angew. Chem., Int. Ed. Engl. 1999, 38, 2411-
2413.
(46) Muci, A. R.; Buchwald, S. L. Top. Curr. Chem. 2002, 219, 131-209.
(47) Wolfe, J. P.; Tomori, H.; Sadighi, J. P.; Yin, J. J.; Buchwald, S. L. J. Org.
Chem. 2000, 65, 1158-1174.
(48) Zapf, A.; Ehrentraut, A.; Beller, M. Angew. Chem., Int. Ed. 2000, 39, 4153-
4155.
(49) Ehrentraut, A.; Zapf, A.; Beller, M. J. Mol. Catal. A 2002, 515-523.
(50) Grasa, G. A.; Viciu, M. S.; Huang, J. K.; Nolan, S. P. J. Org. Chem. 2001,
66, 7729-7737.
Trends from Reaction of 3 with 7. Figure 7 shows the
structures of the ligands that consistently generated catalysts
that formed coupled product in yields greater than 50% from
the test substrates. With the exception of L100, the phosphine
ligands in catalysts that produced a higher than 50% yield
contained one or two tert-butyl groups. These results are in
(51) Viciu, M. S.; Germaneau, R. F.; Nolan, S. P. Org. Lett. 2002, 4, 4053-
4056.
(52) Huang, J.; Nolan, S. J. Am. Chem. Soc. 1999, 121, 9889-9890.
(53) Huang, J.; Grasa, G.; Nolan, S. P. Org. Lett. 1999, 1, 1307-1309.
(54) Zhang, C.; Huang, J.; Trudell, M. L.; Nolan, S. P. J. Org. Chem. 1999, 64,
3804-3805.
(55) Kataoka, N.; Shelby, Q.; Stambuli, J. P.; Hartwig, J. F. J. Org. Chem. 2002,
67, 5553-5566.
(41) Stauffer, S. R.; Lee, S.; Stambuli, J. P.; Hauck, S.; Hartwig, J. F. Org.
Lett. 2000, 9, 1423-1426.
(56) Kranenburg, M.; van der Burgt, Y. E. M.; Kamer, P. C. J.; van Leeuwen,
P. W. N. M.; Goubitz, K.; Fraanje, J. Organometallics 1995, 14, 3081-
3089.
(42) Glas, H.; Herdtweck, E.; Spiegler, M.; Pleier, A.-K.; Thiel, W. R. J.
Organomet. Chem. 2001, 626, 100-105.
(57) Driver, M. S.; Hartwig, J. F. J. Am. Chem. Soc. 1996, 118, 7217-7218.
(58) Room-temperature aminations of aryl iodides have been reported: Wolfe,
J. P.; Buchwald, S. L. J. Org. Chem. 1997, 62, 6066-6068.
(43) Taylor, J. R. An Introduction to Error Analysis, 2nd ed.; University Science
Books: Sausalito, CA, 1997.
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Figure 7. Ligands that gave >50% yield for the coupling of 3 with 7.
reproducibly higher than 50%, as determined by the fluorescence
measurement. The structures of these two ligands were much
different from others shown to be active by a combination of
this assay and more conventional methods.46,59-64 Thus, we
tested the activity of L59 and L106 for the related reaction of
morpholine with bromotoluene. The reaction conducted with
L59 gave 12% yield, and the reaction conducted with L106 gave
no product after 16 h at room temperature. We do not have a
firm explanation for the high yields determined by fluorescence
with these ligands, but reactions conducted with bisimine ligand
L106 were black in color. This ligand is poised for aldol
chemistry, which could generate unsaturated polymers that
would either absorb the incident light or quench the free amine
fluorescence.
Reaction of Dansylpiperazine 3 with Choroarene Dye 9.
We also tested the coupling of amine 3 with chloroarene 9 at
70 °C with catalysts derived from the library of ligands. Again,
a 96-well plate was loaded sequentially with ligand (3.0 mol %
for monodentate and 1.5 mol % for bidentate ligands), fluoro-
phore (3, 7.5 µmol), chlorodye 9 (7.5 µmol), NaOt-Bu, and
CpPd(allyl) or Pd(OAc)2 (3 mol %). The reactions were
conducted in duplicate at 70 °C for 12 h with ligands L1-
L119. An aliquot was removed from each well after 12 h of
reaction, and the aliquot was diluted to 10-5 M. The percent
yield of product was determined as described previously. Figure
8 shows the average percent yields for the aminations of
chloroarene 9 with catalysts bearing the 119 ligands.
Figure 8. Yields determined by fluorescence measurements for the coupling
of 3 with 9 in the presence of palladium catalysts containing 119 different
ligands.
than 20% yield. All reactions that occurred in yields by
fluorescence greater than 30% showed a percent uncertainty less
than 30. The 23 reactions that occurred in yields greater than
50% in one or two of the runs showed a percent uncertainty
between 1 and 12%. The average uncertainty of the yields from
these reactions was just 5%.
Trends from Reactions of 3 with 9. The structures of the
23 ligands that generated product in greater than 50% yield are
shown in Figure 9. The carbene ligands provided the most active
systems for the coupling of aryl chlorides under the conditions
of the screen. The pentaphenylferrocenyl ligands (Q-phos) L17-
L19 were the next most active. Some trends could again be
assigned for the relative activities of the heterocyclic carbene
ligands. Catalysts generated from the dihydroimidazolium salts
L115 and L116 were more active for reactions of aryl chlorides
than were those generated from the imidazolium salts. This trend
was confirmed by subsequent studies in our labs41 and has been
published recently by Nolan.50 In contrast to reactions of
bromoarenes at room temperature catalyzed by complexes of
the three Q-phos derivatives, FRET results indicated that the
three Q-phos derivatives L17, L18, and L19 generated catalysts
with nearly equal activity toward chloroarene 9.
The variations in yields by fluorescence for the two runs with
chloroarene 9 were similar to the variations with bromoarene
7. Only a few reactions showed significant variability in yields
for the two experiments, and these reactions occurred in less
(59) Belfield, A. J.; Brown, G. R.; Foubister, A. J. Tetrahedron 1999, 55, 11399-
11428.
(60) Hartwig, J. F. Angew. Chem., Int. Ed. 1998, 37, 2046-2067.
(61) Hartwig, J. F. In Modern Amination Methods; Ricci, A., Ed.; Wiley-VCH:
Weinheim, Germany, 2000.
(62) Kingsbury, C. L.; Mehrman, S. J.; Takacs, J. M. Curr. Org. Chem. 1999,
3, 497-555.
(63) Wolfe, J. P.; Wagaw, S.; Marcoux, J.-F.; Buchwald, S. L. Acc. Chem. Res.
1998, 31, 805-818.
(64) Yang, B. H.; Buchwald, S. L. J. Organomet. Chem. 1999, 576, 125-146.
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FRET as a High-Throughput Assay for Coupling Reactions
A R T I C L E S
Table 1. Comparison of FRET Data for the Amination of Bromo
and Chloroarene Dyes with the 30 Ligands that Generated the
Most Active Catalysts for Reactions of 3 with 7 and 9a
a Ligands in bold indicate those generating above 50% yield. Ligands
in red were used in the screen for reactions in polar solvents with K3PO4
as base (see text).
low activity of palladium complexes of aryl phosphines as
catalysts for the reactions of aryl chlorides and the accuracy of
the FRET method when screening a library of ligands.
Investigation of Potential False Negatives. Reactions of the
bromo- and chlorodyes with catalysts containing certain ligands,
particularly L4, L21, L27, and L69, occurred in yields by the
fluorescence analysis that were lower than those reported
previously for reactions of related unlabeled substrates. Catalysts
containing these four ligands are known to couple electron
neutral aryl chlorides and bromides with cyclic secondary
amines in yields greater than 80%.29,65 To confirm that these
ligands provide high yields for related substrates under the
conditions of the FRET assay, we evaluated the reaction of
bromotoluene with morpholine in the presence of (allyl)PdCp
and (t-Bu)3P (L69). As expected, N-tolylmorpholine was formed
in 98% yield by GC yield after 16 h.
To probe the origin of these lower yields in the FRET assay,
we evaluated whether the method for assembly of reactions,
the method of analysis, or the type of substrate created the
moderate yields. Initially, we tested whether an inaccuracy in
the Pd/L ratio from assembly of small-scale reactions was
contributing to the low yields;29,66 however, experiments with
2.8 or 3.0 mol % ligand and 3.0 mol % (allyl)PdCp added
carefully from stock solutions generated no significant difference
in the yields of coupled product formed from dyes 3 and 7 in
the presence of (allyl)PdCp and any of the four ligands. In
addition, increasing the amount of catalyst to 6 mol % (allyl)-
PdCp and P(t-Bu)3 and allowing the reaction to occur over
several days did not increase the yield, as determined by FRET.
Thus, we conducted the three reactions in Scheme 3. First,
we determined by 1H NMR spectroscopy the yield for the
coupling of 3 with 7 in the presence of 3 mol % CpPd(allyl)
and P(t-Bu)3 in C6D6 solvent. This reaction produced the coupled
product in 60% yield, and this yield is consistent with those
determined by FRET. To evaluate whether halide 7 or amine 3
caused the yields to be lower than those observed with simpler
Figure 9. Ligands that gave >50% yield for reaction of 3 with 9.
Comparison of Reactions of 3 and 7 with Reaction of 3
with 9. Catalysts derived from ligands in the library coupled
bromo- and chloroarenes in different yields. The results in Table
1 indicate that some ligands created catalysts that formed
coupled product from the chloroarene in higher yields than from
the bromoarene. For example, carbene ligand precursor L114,
which generated catalysts with only modest activity toward the
reaction of bromoarene 7 at room temperature (45%), showed
good activity toward the reaction of chlorodye 9 at 70 °C. In
addition, reactions conducted with ligands L29 and L64 both
occurred in slightly higher yields with chloroarene 9 than with
bromoarene 7. These higher yields for the reaction of the
chloride 9 in the presence of these catalysts is likely a result of
the higher temperatures at which the reactions of chloroarenes
were conducted. If catalysts formed from L29 and L64 react
cleanly with both aryl bromides and chlorides, but reactions
with these substrates occur at similar rates and require elevated
temperatures, then yields from the reaction of chloride 9 would
be higher than yields from the reaction of bromide 7.
Table 1 also reveals the expected finding that several ligands,
such as L27, L38, and L100, are more active for the amination
of aryl bromides than for the amination of chlorides. The
difference in activity of catalysts generated from Xantphos L100
for reactions of aryl bromides and chlorides underscores the
(65) Wolfe, J. P.; Buchwald, S. L. Angew. Chem., Int. Ed. 1999, 38, 2413-
2416.
(66) The difficulty in controlling the metal-to-ligand ratio when handling small
quantities of solids was noted previously in ref 1.
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A R T I C L E S
Stauffer and Hartwig
Scheme 3
substrates, the reaction of morpholine with bromodye 7 in C6D6
for 14 h was conducted. This reaction occurred in 64% isolated
yield after chromatography (Scheme 3, middle). In contrast, the
reaction of fluorescent amine 3 and bromotoluene (Scheme 3,
bottom) formed the N-tolylamine in 95% isolated yield. Thus,
the yield of coupled product from the reaction of 3 with 7
determined by fluorescence was accurate, and the reaction of
the haloarene tethered to the azodye quencher catalyzed by some
of the metal ligand combinations occurred in lower yields than
reactions of less functionalized aryl halides.
During previous studies, yields from reactions of acrylates
(Heck couplings)20 and cyanoesters19 with the haloarene azodyes
catalyzed by palladium complexes of P(t-Bu)3 were similar to
those with less functionalized haloarene substrates. These
previous results contrast with results in the current study with
some of the catalysts. The difference in the strength of the base
between the previous studies and the current work may account
for these contrasting results. Phosphate and amine bases were
used in the previous studies, while NaOt-Bu base was used in
the current work.
was approximately 17% of the total volume of the reactions.
Six different polar solvents and solvent mixtures were tested,
and CpPd(allyl) was used as the source of palladium. Fifteen
ligands that generated the most active catalysts in the coupling
of the bromo- and chlorodye were chosen to screen for activity
in the presence of K3PO4 as base.
The results from this experiment are shown in Figure 10. A
mixture of dioxane and m-xylene with K3PO4 as base was
included to compare results from reactions with this base to
those in a similar medium with NaOt-Bu as base described
earlier. In addition, DME, NMP, DMSO, DMA, and a mixture
of t-BuOH and m-xylene were tested as solvent. Reactions in
DMSO formed an intractable tar, and the results from these
reactions are not shown. Figure 10 shows that reactions
catalyzed by palladium and certain ligands occurred in high yield
in the presence of K3PO4 when conducted in the appropriate
solvent mixtures. Three of these ligands, L4, L27, and L69,
generated complexes that catalyzed reactions of the azodye in
mixtues of dioxane and xylene in much higher yield than when
NaO-t-Bu was used as base in this medium. These higher yields
from reactions conducted with K3PO4 agree with our conclusion
that the yields for reactions with the azodye quencher and
NaO-t-Bu as base differ from yields with other aryl bromides
and from yields obtained with the FRET assay during previous
studies with phosphate or amine as base19,20 because of the
difference in strength of the base.
Reactions of Amine 3 with Bromoarene 7 in Polar Solvents
with K3PO4 as Base. A final set of reactions was performed at
80 °C with bromodye 7, fluorophore 3, and the milder K3PO4
base for three reasons: to evaluate if the strong base led to the
lower yields with the azodye, to develop conditions for
aminations in more polar solvents, and to demonstrate the utility
of screening to find a new medium for a reaction. This base
was delivered as an aqueous solution, and this aqueous solution
None of the ligands gave high yields in the most polar
solvents. However, several ligands gave high yields in mixtures
of xylene and tert-butyl alcohol. Reactions catalyzed by
complexes of 8 of the 15 ligands (L4, L5, L18, L27, L69, L73,
L75, and L76) gave yields over 70% in this medium, and
ferrocenyl ligands L14 and L19 and trialkylphosphines L69 and
L73 gave yields in the range of 80%. Thus, reactions in mixtures
of polar and nonpolar organic solvents can be appropriate for
the amination chemistry.67
Summary
Overall, this work shows that FRET can be used to screen
homogeneous catalysts for the amination of aryl halides and,
(67) The ability to conduct reactions with a two-phase mixture of toluene and
water with hydroxide base in the presence of a phase-transfer agent was
demonstrated recently but not with polar substrates that are insoluble in
the nonpolar toluene: Kuwano, R.; Utsunomiya, M.; Hartwig, J. F. J. Org.
Chem. 2002, 67, 6479-6486.
Figure 10. Yields determined by FRET for the reaction of 3 with 7 at 80
°C for 16 h in the presence of K3PO4 as base.
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FRET as a High-Throughput Assay for Coupling Reactions
A R T I C L E S
by analogy, to screen catalysts for other processes. The study
reported here evaluated catalysts derived from a library of
phosphine and heterocyclic carbene ligands for two substrate
combinations in several media. The reaction yields obtained by
the FRET method were reproducible and agreed in most cases
with yields obtained by conventional methods. Some differences
in yields were observed between the dye substrates and
unfunctionalized substrates, but only with a small subset of the
ligands. As a test of the FRET method to evaluate new reaction
media, conditions to conduct the amination chemistry with K3-
PO4 in polar solvent mixtures were developed.
01) for postdoctoral fellowship support. We thank Kevin
Shaughnessy for initial fluorescent studies and James Stambuli,
Sunwoo Lee, and Quinetta Shelby for providing several of the
phosphine ligands.
Supporting Information Available: Full Experimental Sec-
tion including the structures and synthetic methods for prepara-
tion of ligands used in the screening assay and procedures for
all catalyst screening (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
Acknowledgment. We thank the NIH-NIGMS (GM55382)
for support of this work, and S.R.S. thanks NIH (GM-20191-
JA034161P
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